Fluorophore Labeling of Native FKBP12 by Ligand-Directed Tosyl Chemistry Allows Detection of Its Molecular Interactions in Vitro and in Living Cells
Fluorophore Labeling of Native FKBP12 by Ligand-Directed Tosyl Chemistry Allows Detection of Its Molecular Interactions in Vitro and in Living Cells
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DOI：
10.1021/ja401956b
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发表时间：
2013-05-08
影响因子：
15
通讯作者：
Hamachi, Itaru
中科院分区：
文献类型：
--
作者：
Tamura, Tomonori;Kioi, Yoshiyuki;Hamachi, Itaru
将合成的荧光团引入特定的内源性蛋白质并分析其在活细胞中的功能是化学生物学的一个巨大挑战。为此，我们证明了天然FKBP 12（FK 506结合蛋白12）在体外和活细胞中使用配体导向甲苯磺酰基（LDT）化学的靶向选择性和位点特异性荧光标记。LDT介导的标记产生在催化口袋附近含有俄勒冈州绿色（OG）染料的半合成FKBP 12。OG-FKBP 12作为一种荧光报告分子，可以在体外监测其与雷帕霉素和FRB（FKBP-rapamycin-binding domain）的相互作用。我们还成功地证明了通过结合使用荧光蛋白标签技术和福斯特共振能量转移成像，在活细胞内雷帕霉素介导的OG-FKBP 12和FRB复合的可视化。
Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent proteintag technology and Forster resonance energy-transfer imaging.